The present invention relates to live attenuated bacteria for use in a medicament, to vaccines based upon such bacteria useful for the prevention of microbial pathogenesis, to live attenuated bacteria carrying a heterologous gene and to methods for the preparation of such vaccines and bacteria.
The means by which a warm blooded animal overcomes microbial pathogenesis is a complex process. Immunity to microbial pathogenesis is one means by which a warm blooded animal avoids pathogenesis, or suffers a less intense pathogenic state. Incomplete immunity to a given pathogen results in morbidity and mortality in a population exposed to a pathogen. It is generally agreed that vaccines based on live but attenuated micro-organisms (live attenuated vaccines) induce a highly effective type of immune response. Such vaccines have the advantage that, once the animal host has been vaccinated, entry of the microbial pathogen into the host induces an accelerated recall of earlier, cell-mediated or humoral immunity which is able to control the further growth of the organism before the infection can assume clinically significant proportions. Vaccines based on a killed pathogen (killed vaccine) are generally conceded to be unable to achieve this type of response. However, vaccines that contain a live pathogen present, depending on the level of attenuation, the danger that the vaccinated host upon vaccination may contract the disease against which protection is being sought. Therefore, it would be desirable to have a vaccine that possesses the immunising attributes of a live micro-organism but that is not capable of causing undesirable side effects upon vaccination.
The general approach for attenuating bacteria is the removal of one or more virulence factors. In most cases however, virulence factors also play a role in inducing immunity. In those cases, deletion of virulence factors unavoidably impairs the immunogenic capacities of the bacterium. This is of course an unwanted situation. A live vaccine should preferably retain the antigenic complement of the wild-type strain. Moreover, the live vaccine should be sufficiently a-virulent to avoid unacceptable pathological effects, but on the other hand it must cause a sufficient level of immunity in the host. Finally, the live attenuated vaccine strain should preferably have substantially no probability for reverting to a virulent wild type strain.
It was now surprisingly found that a gene encoding a protein known to play a role in the central carbohydrate metabolism in many bacterial genera can be deleted, causing attenuated behaviour in vivo without impairing the viability of such bacteria in vivo. Bacteria from which this gene is deleted do unexpectedly show an attenuated character. Moreover, since the encoded protein plays no role in the induction of immunity, the antigenic load of bacteria from which this gene is deleted, is identical to that of the wild-type. Therefore, such bacteria could unexpectedly be advantageously used in the field of preparation of medicaments, more specifically for the preparation of live attenuated vaccines. The gene that according to the present invention can be deleted and leads to an attenuated in vivo behaviour of the deletion mutants is a gene formerly known as the fruR gene, currently however called the cra gene. It was known that mutants lacking this gene could be grown in vitro, but only if the deficiencies due to lack of Cra activity are compensated for in the growth medium. This means that nutrients on which the Cra-deficient mutant can grow must be present in the growth medium. Generally spoken, pathogenic bacteria are self-supporting in the sense that they adapt their metabolism to the nutrients that are available. The cra gene plays such an adaptive role in many main metabolic pathways (see below). Mutants from which the cra gene has been deleted can however grow perfectly well on glucose and many other sugars as carbon source. In the host animal, such sugars are available and therefore one would not expect the cra gene to be functional under in vivo conditions. And thus, one would not expect Cra-negative mutants to show attenuated characteristics in the host. That explains why, although such mutants were known in the art, they have never been suggested to be potential live attenuated vaccine candidates.
One embodiment of the invention relates to live attenuated bacteria that are no longer capable to express a functional Cra protein as a result of a mutation in the cra gene, for use in a vaccine.
The gene product, (formerly known as FruR; the Fructose Repressor Protein), now also known as Cra (the Catabolite Repressor/Activator Protein), is a regulatory protein in many main pathways of the carbohydrate metabolism.
The cra-gene product Cra regulates the central carbon metabolism. More specifically, Cra positively regulates transcription of genes encoding biosynthetic and oxidative enzymes (e.g. key enzymes in the TCA cycle, the glyoxalate shunt, the gluconeogenic pathway and electron transport) by binding upstream of the promoters of these genes and negatively regulates transcription of genes encoding glycolytic enzymes (e.g. key enzymes in the Embden-Meyerhof and Entner-Doudoroff pathways). Due to its key position in carbohydrate metabolism, the cra gene and its gene product Cra are widespread in the bacterial realm. The Cra protein is a highly conserved protein. It can be found in e.g. Escherichia coli, in Salmonella enterica species, such as serotype Typhimurium, Enteritidis and Dublin, in Actinobacillus species such as A. pleuropneumoniae, in Haemophilus species such as H. paragallinarum, in Aeromonas salmonicidae, in Pasteurella species such as P. piscida and P. multocida, in Streptococcus species such as S. equi and S. suis and in Yersinia species such as Y. pestis. 
The gene itself and its complete nucleotide sequence in Salmonella and Escherichia have been elucidated already in 1991 by Jahreis, K. et al. (Mol. Gen. Genet. 226: 332-336 (1991)). Jahreis showed that the Cra protein in Salmonella enterica, serotype Typhimurium and Escherichia coli differed only in 4 positions, of which two were merely conservative exchanges. This is of course in line with what could be expected for a protein playing a role in so many universal pathways in the bacterial carbohydrate metabolism, especially where E. coli and Salmonella diverged not that far during evolution. The mechanism of binding of the Cra protein has been at least partially elucidated by Ramseier, T. M. et al. (J. Mol. Biol. 234:28-44 (1993)). The role and function of the Cra protein (the Catabolite Repressor/Activator Protein) have been regularly described in the literature, e.g. in a recent mini-review by Saier, M. H. and Ramseier, T. M. (Journ. Bacteriol. 178: 3411-3417 (1996)).
Such a mutation can be an insertion, a deletion, a substitution or a combination thereof, provided that the mutation leads to the failure to express a functional Cra protein. A functional Cra protein is understood to be a protein having the regulating characteristics of the wild-type protein. Therefore, a Cra protein that is defective in at least one of its functions is considered to be a non-functional Cra protein.
Live attenuated bacteria for use according to the invention can be obtained in several ways. One possible way of obtaining such bacteria is by means of classical methods such as the treatment of wild-type bacteria having the cra gene with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment. Strains that do not produce a functional Cra protein can easily be picked up. They grow on minimal medium exclusively in the presence of glucose and other sugars as carbon sources (which differentiates them from cya and crp mutants) but they are not able to grow with gluconeogenic substrates as sole carbon source. (Chin et al., J. Bacteriol. 169: 897-899 (1987)) They can therefore very easily be selected in vitro. The nature of the mutation caused by classical mutation techniques is unknown. This may be a point mutation which may, although this is unlikely to happen, eventually revert to wild-type. In order to avoid this small risk, transposon mutagenesis would be a good alternative. Mutagenesis by transposon mutagenesis, is also a mutagenesis-technique well-known in the art. This is a mutation accomplished at a localised site in the chromosome. Transposon-insertions can not be targeted to a specific gene. It is however very easy to pick up cra-mutants since they do not grow in vitro without nutrient compensation for lack of Cra activity. Therefore, they can easily be selected from a pool of randomly transposon-mutated bacteria. A possibility to introduce a mutation at a predetermined site, rather deliberately than randomly, is offered by recombinant DNA-technology. Such a mutation may be an insertion, a deletion, a replacement of one nucleotide by another one or a combination thereof, with the only proviso that the mutated gene no longer encodes functional Cra. Such a mutation can e.g. be made by deletion of a number of nucleic acids. Even very small deletions such a stretches of 10 nucleic acids can already render Cra non-functional. Even the deletion of one single nucleic acid may already lead to a non-functional Cra, since as a result of such a mutation, the other nucleic acids are no longer in the correct reading frame. Each deletion of insertion of a number of nucleic acids indivisible by three causes such a frame shift. More preferably, a longer stretch is removed e.g. 100 nucleic acids. Even more preferably, the whole cra gene is deleted. It can easily be seen, that especially mutations introducing a stop-codon in the open reading frame, or mutations causing a frame-shift in the open reading frame are very suitable to obtain a strain which no longer encodes functional Cra.
All techniques for the construction of Cra-negative mutants are well-known standard techniques. They relate to cloning of the Cra-gene, modification of the gene sequence by site-directed mutagenesis, restriction enzyme digestion followed by re-ligation or PCR-approaches and to subsequent replacement of the wild type cra gene with the mutant gene (allelic exchange or allelic replacement). Standard recombinant DNA techniques such as cloning the cra gene in a plasmid, digestion of the gene with a restriction enzyme, followed by endonuclease treatment, re-ligation and homologous recombination in the host strain, are all known in the art and described i.a. in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309-6). Site-directed mutations can e.g. be made by means of in vitro site directed mutagenesis using the Transformer(copyright) kit sold by Clontech. PCR-techniques are extensively described in (Dieffenbach and Dreksler; PCR primers, a laboratory manual. ISBN 0-87969-447-3 and ISBN 0-87969447-5).
The cra gene comprises not only the coding sequence encoding the Cra protein, but also regulatory sequences such as the promoter. The gene also comprises sites essential for correct translation of the Cra mRNA, such as the ribosome binding site. Therefore, not only mutations in the coding regions but also mutations in those sequences essential for correct transcription and translation are considered to fall within the scope of the invention.
In a preferred embodiment, the invention relates to live attenuated bacteria of the genera Escherichia, Salmonella, Actinobacillus, Haemophilus, Aeromonas, Pasteurella, Streptococcus and Yersinia for use in a vaccine.
In a more preferred form of the invention, the live attenuated bacterium according to the invention is selected from the group consisting of S. enterica serotype Typhimurium, Enteritidis, Choleraesuis, Dublin, Typhi, Gallinarum, Abortusovi, Abortus-equi, Pullorum, E. coli or Y. pestis. These bacterial genera comprise a large number of species that are pathogenic to both humans and a variety of different animals.
In an even more preferred form thereof, the live attenuated bacterium according to the invention is S. enterica, E. coli or Y. pestis. 
In a still even more preferred form, this embodiment relates to live attenuated bacteria according to the invention in which the mutation in the Cra gene has been made by recombinant DNA technology.
Well-defined and deliberately made mutations involving the deletion of fragments of the cra gene or even the whole gene or the insertion of heterologous DNA-fragments or both, have the advantage, in comparison to classically induced mutations, that they will not revert to the wild-type situation. Thus, in an even more preferred form, this embodiment of the invention refers to live attenuated bacteria in which the cra gene comprises an insertion and/or a deletion.
Given the large amount of vaccines given nowadays to both pets and farm animals, it is clear that combined administration of several vaccines would be desirable, if only for reasons of decreased vaccination costs. It is therefore very attractive to use live attenuated bacteria as a recombinant carrier for heterologous genes, encoding antigens selected from other pathogenic micro-organisms or viruses. Administration of such a recombinant carrier has the advantage that immunity is induced against two or more diseases at the same time. The live attenuated bacteria for use in a vaccine, according to the present invention provide very suitable carriers for heterologous genes, since the gene encoding the Cra protein can be used as an insertion site for such heterologous genes. The use of the cra gene as an insertion site has the advantage that at the same time the cra gene is inactivated and the newly introduced heterologous gene can be expressed (in concert with the homologous bacterial genes). The construction of such recombinant carriers can be done routinely, using standard molecular biology techniques such as allelic exchange. Therefore, another embodiment of the invention relates to live attenuated recombinant bacteria, preferably of the genera Escherichia, Salmonella, Actinobacillus, Haemophilus, Aeromonas, Pasteurella, Streptococcus and Yersinia that do not produce a functional Cra protein, and in which a heterologous gene is inserted. Such a heterologous gene can, as mentioned above, e.g. be a gene encoding an antigen selected from other pathogenic micro-organisms or viruses. Such genes can e.g. be derived from pathogenic herpes viruses (e.g. the genes encoding the structural proteins of herpes viruses), retro viruses (e.g. the gp160 envelope protein), adenoviruses and the like. Also a heterologous gene can be obtained from pathogenic bacteria. As an example, genes encoding bacterial toxins such as Actinobacillus pleuropneumoniae toxins, Clostridium toxins, outer membrane proteins and the like are very suitable bacterial heterologous genes. Another possibility is to insert a gene encoding a protein involved in triggering the immune system, such as an interleukin or an interferon, or another gene involved in immune-regulation.
Insertion of the heterologous gene in the cra gene is advantageous, since there is no need to find an insertion site for the heterologous gene, and at the same time the cra gene is knocked out. Thus, in a preferred form of this embodiment the heterologous gene is inserted in the cra gene. The heterologous gene can be inserted somewhere in the cra gene or it can be inserted at the site of the cra gene while this gene has been partially or completely deleted.
Because of their unexpected attenuated but immunogenic character in vivo, the bacteria for use in a vaccine, according to the invention are very suitable as a basis for live attenuated vaccines. Thus, still another embodiment of the invention relates to live attenuated vaccines for the protection of animals and humans against infection with a bacterium of which the wild type form comprises a cra gene. Such vaccines comprise an immunogenically effective amount of a live attenuated bacterium for use in a vaccine, according to the invention or a live recombinant carrier bacterium according to the invention, and a pharmaceutically acceptable carrier.
Preferably, the vaccine comprises a live attenuated bacterium according to the invention, selected from the group of Escherichia, Salmonella, Actinobacillus, Haemophilus, Aeromonas, Pasteurella, Streptococcus and Yersinia. Immunogenically effective means that the amount of live attenuated bacteria administered at vaccination is sufficient to induce in the host an effective immune response against virulent forms of the bacterium.
In addition to an immunogenically effective amount of the live attenuated bacterium described above, a vaccine according to the present invention also contains a pharmaceutically acceptable carrier. Such a carrier may be as simple as water, but it may e.g. also comprise culture fluid in which the bacteria were cultured. Another suitable carrier is e.g. a solution of physiological salt concentration.
The useful dosage to be administered will vary depending on the age, weight and animal vaccinated, the mode of administration and the type of pathogen against which vaccination is sought.
The vaccine may comprise any dose of bacteria, sufficient to evoke an immune response. Doses ranging between 103 and 1010 bacteria are e.g. very suitable doses.
Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Adjuvants are non-specific stimulators of the immune system. They enhance the immune response of the host to the vaccine. Examples of adjuvants known in the art are Freunds Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, cf. for instance European Patent EP 109942), Saponins, mineral oil, vegetable oil, and Carbopol. Adjuvants, specially suitable for mucosal application are e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT). Other suitable adjuvants are for example aluminium hydroxide, aluminium phosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F(copyright) or Marcol 52(copyright), saponins or vitamin-E solubilisate.
Therefore, in a preferred form, the vaccines according to the present invention comprise an adjuvant.
Other examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. phosphate buffer). Especially when such stabilisers are added to the vaccine, the vaccine is very suitable for freeze-drying. Therefore, in a more preferred form, the vaccine is in a freeze-dried form.
For administration to animals or humans, the vaccine according to the present invention can be given inter alia intranasally, intradermally, subcutaneously, orally, by aerosol or intramuscularly. For application to poultry, wing web and eye-drop administration are very suitable.
Still another embodiment relates to the use of a bacterium for use in a vaccine or a recombinant bacterium according to the invention for the manufacture of a vaccine for the protection of animals and humans against infection with a wild type bacterium or the pathogenic effects of infection.
Still another embodiment of the invention relates to methods for the preparation of a vaccine according to the invention. Such methods comprise the admixing of a live attenuated bacterium according to the invention or a live recombinant carder bacterium according to the invention, and a pharmaceutically acceptable carrier.